Introduction

Down syndrome (DS, [1]) is the leading genetic cause of intellectual disability and the most common chromosomal disorder compatible with human survival [2], [3], [4], [5]. DS affects approximately 1 in 700 live births and an estimated 220,000 newborns each year [6]. In the majority of cases, the disorder arises from sporadic non-disjunction of chromosome 21 (HSA21) and triplication of the entire chromosome [7], [8], [9], or infrequently, from partial aneuploidy due to unbalanced chromosomal translocation [10], [11], [12], [13]. In most cases, DS aneuploidy disrupts gene-dose equilibrium in all somatic cells, whereas a minority of cases demonstrates mosaicism [14]. Chromosomal triplication invariably includes the APP gene (21q21) that encodes the amyloid precursor protein, APP [15], [16]. Endoproteolytic cleavage of APP yields the pathogenic amyloid-β peptides (Aβ) that progressively accumulate in the brain as the diffuse and neuritic plaques of Alzheimer's disease (AD). In DS, cerebral Aβ accumulation is greatly accelerated [17] and leads to invariant early-onset AD neuropathology and age-dependent neurocognitive sequelae [18], [19].

Lens abnormalities in subjects with clinical features of DS were first reported over a century ago [20], [21] followed by numerous confirmatory reports since [20], [22], [23], [24]. The distinctive DS lens phenotype clinically manifests as cerulean “blue dot” opacities that often emerge in the first decade of life, and in some cases, may be evident at birth [22], [25], [26], [27]. DS cataracts typically localize to the supranuclear region and are histologically characterized by granular material of uncertain composition [20], [24], [25]. The molecular origin and pathogenic mechanisms by which the DS aneuploidy is expressed as a distinctive age-dependent lens phenotype are unknown.

We previously reported disease-linked supranuclear cataracts that correlate with pathogenic Aβ accumulation, classical amyloid pathology, and co-localizing pathology in lenses obtained from subjects with Alzheimer's disease (AD) but not in those with other neurodegenerative disorders nor in normal aged controls [28]. In AD lenses, Aβ accumulates as electron-dense cytosolic aggregates (longest axial dimension, ∼5 nm to ∼200 nm) that distribute heterogeneously within the cytoplasm of supranuclear and deep cortical lens fiber cells. AD-linked Aβ aggregates in the lens qualify as Raleigh scattering centers that clinically manifest as supranuclear opacities that ultimately progress to frank cataracts. These AD-linked supranuclear cataracts are phenotypically, anatomically, ultrastructurally, and biochemically distinguishable from common age-related nuclear cataracts. Given the association between Aβ amyloid lens pathology in AD [28], we hypothesized that subjects with DS would also demonstrate age-related Aβ amyloid pathology in the lens. The aim of the present study was to test the hypothesis that DS-linked lens pathology reflects accelerated Aβ accumulation and co-localizing amyloid pathology that clinically manifests as the characteristic supranuclear cataract phenotype associated with this common chromosomal disorder.

Ethical review and permission to conduct this investigation were approved by Harvard Medical School, Brigham and Women's Hospital, Massachusetts Eye and Ear Infirmary, and Children's Hospital Boston. This study conforms to applicable regulatory guidelines at Harvard Medical School and principles of human research subject protection in the Declaration of Helsinki. Patients were informed, consent forms signed, and patient samples collected and signed in accordance with an approved IRB protocol (02-08-053X) at the Massachusetts Eye and Ear Infirmary, Boston, MA, USA.

(A) Characteristic circumferential supranuclear cataract in the lens of a 64-year-old male subject with Down syndrome. This distinctive cataract is evident as an annular half-toroid band of opacification in the deep cortical and supranuclear subregions of the lens. This same lens specimen is presented as a slit lamp biomicrocopic image (Fig. 1H) and as a stereo image pair (with intact zonule fibers, Fig. S1). This dramatic Down syndrome cataract is phenotypically comparable to the incomplete subequatorial supranuclear cataract observed in the lens of a 76-year-old male subject with advanced Alzheimer's disease (B). These distinctive supranuclear cataracts are not observed in age-normal control subjects. See text for details.

A representative stereo image of a lens from a 64-year-old male with DS (Fig. 2A) illustrates an advanced circumferential supranuclear cataract characteristic of the DS phenotype. The phenotypic features of this distinctive cataract phenotype are demonstrated in the same lens observed by slit lamp photomicroscopy (Fig. 1H) and as a stereo image pair with intact zonule fibers (Fig. S1). The cataract phenotype observed in DS was strikingly similar to the mature subequatorial supranuclear cataracts previously reported in advanced AD (Fig. 2B;[28]). This distinctive DS-associated cataract was not present in age-matched normal control subjects nor was this focal lens pathology associated with global swelling, diffuse opacification, or capsular disruption indicative of postmortem tissue injury.

Histochemical and Ultrastructural Analyses

The phenotypic correspondence of the distinctive cataracts in DS and AD encouraged us to investigate the possibility of Aβ pathology in DS lenses. Orientation to the anatomy of the adult human lens is shown (Fig. 3A,B) and compared to an historical drawing (Fig. 3C) illustrating the distinctive localized supranuclear lens pathology in a 37-year-old subject with clinical features consistent with Down syndrome [23]. Amyloid histochemical analysis of DS lenses in the present study demonstrated Congo red staining (Fig. 3D) and intense apple-green birefringence (Fig. 3E) under cross-polarized illumination, findings consistent with the tinctorial requirements for designation as amyloid pathology [30], [31]. Amyloid histopathology was not evident in a lens from an age- and sex-matched normal control donor (Fig. 3F) and co-localized with lens pathology and Aβ immunoreactivity as demonstrated by specific Aβ immunoreactivity in the deep cortical, supranuclear, and anterior epithelial subregions (Fig. 3G). Anti-Aβ immunoreactivity was not detected in adjacent control sections probed with immunodepleted detection antibody (Fig. 3H) or in lens specimens from age- and sex-matched normal control donors (Fig. 3I). Anti-Aβ immunogold electron microscopy revealed immunoreactive aggregates (∼5–50 nm) that localized to the cytoplasm of supranuclear fiber cells in DS lenses (Fig. 3J,K) but was not observed in adjacent control sections probed with immunodepleted detection antibody (Fig. 3L). These histopathological findings are identical to those previously described for lens pathology in late-onset sporadic AD [28].

Identification and Biochemical Characterization of Aβ in Down Syndrome Lens

Definitive identification of human Aβ in DS lens was accomplished by tryptic digest tandem mass spectrometry peptide sequencing. Biochemical sequencing analysis was conducted on small molecular weight eluates derived from HPLC fractionation of human DS lens protein extract (Fig. 4). In positive electrospray mode, the LC-MS/MS spectrum detected two tryptic peptide signals corresponding to 17LVFFAEDVGSNK28 and 6HDSGYEVHHQK16 that uniquely identify human Aβ. The detected fragments did not derive from the larger amyloid precursor protein (APP) as sequencing was performed on eluates with retention times corresponding to monomeric Aβ (∼4 kDa). In order to establish the tissue concentration of Aβ in the lens, we first investigated the relative efficiency of sodium dodecyl sulfate compared to formic acid extraction performed on protein extracts obtained from human lens homogenates (Fig. 5). Formic acid demonstrated a far superior extraction profile relative to sodium dodecyl sulfate for both Aβ1-40 and Aβ1-42. ELISA results (Fig. 6A,B) demonstrated elevated lens and brain tissue levels of the major human Aβ isoforms (Aβ40 and Aβ42) and total Aβ that were consistently elevated in DS compared to normal controls and comparable to the values previously reported in AD [28]. Immunoblot analysis of DS lens extract (Fig. 6C) revealed a prominent Aβ-immunoreactive band that migrated with an apparent molecular weight of ∼4 kDa corresponding to monomeric Aβ that was also detected in homogenate of AD brain. The apparent Aβ monomer bands detected in DS and AD lens and brain homogenate migrated with the same electrophoretic mobility observed in the single band detected in a sample of purified synthetic human Aβ40. Aβ monomer and apparent oligomeric species were also detected as weak but distinct bands in normal control lens. Total Aβ immunoreactive material detected in the lens of a 2-year-old DS subject was comparable to that detected in lenses from a 57-year-old subject with familial AD and an 85-year-old subject with neuropathologically-confirmed late-onset AD (Fig. 6C). The results of the immunoblot analysis provide confirmatory evidence supporting the finding of Aβ in DS lenses and also demonstrate an apparent increase in oligomeric Aβ and corresponding decrease in monomeric Aβ in the lens as a function of advancing age.

Discussion

In this report, we identify the origin and pathogenic mechanism of cataractogenesis in DS, establish the distinctive lens phenotype in DS as a genetic cataract, and define the molecular correspondence of DS-associated pathology in the lens and brain. Taken together with our previous findings of supranuclear Aβ lens pathology in late-onset sporadic AD [28], these results also establish the pathogenic relationship linking Aβ pathology in the lens and brain in both DS and AD.

AD neuropathology is an invariant feature of DS in subjects over the age of 30 [18], [19] but may emerge as early as the first decade [18], [34]. A critical factor contributing to the association of DS and AD Aβ pathology is the locus of the amyloid precursor protein gene (APP, 21q21) on the long arm of chromosome 21 [16], [35], [36]. Chromosome 21 triplication leads to increased dosage of the APP gene, accelerated cerebral Aβ accumulation, progressive AD neuropathology, and age-dependent cognitive sequelae [18], [37], [38], [39], [40]. With the exception of a single reported case of an individual with an atypical DS karyotype involving a specific chromosome 21 micro-deletion in the APP gene locus [41], all subjects with Down syndrome (including the rare cases involving Robertsonian translocation, partial duplications, or trisomic mosaicism) demonstrate triplication of the APP gene locus (21q21), overexpress amyloid precursor protein (APP) in affected somatic cells, accumulate β-amyloid peptides (Aβ) in the brain, and reveal evidence of early-onset Alzheimer's disease (AD) neuropathology [18], [19], [34], [40], [42].

In this report, we hypothesize and confirm that this genotype-phenotype concordance extends to Aβ-linked molecular pathology in the supranucleus and deep cortex of the lens. The characteristic cataract phenotype associated with Aβ lens pathology has been identified in only two clinical disorders, Alzheimer's disease [28] and as reported here, Down syndrome. This distinctive supranuclear cataract phenotype has not been reported in normal individuals, and is not observed in other non-AD neurodegenerative diseases, nor in normal aged controls [28]. The classical cerulean “blue dot” cataracts characteristic of DS initially emerge at the equatorial periphery of the lens, and over time, encompass progressively larger areas of the supranucleus and deep cortex (Fig. 8). While lens pathology observed in both infantile and mature DS lenses reveal opaque regions that are clinically described as supranuclear, the ages of the corresponding fiber cells comprising these regions are different (Fig 8). In this respect, it is important to note that the lens pathology in DS lenses does not implicate involvement of lens fibers cells in the fetal nucleus.

The anatomical localization of the characteristic Down syndrome cataract phenotype (white shading) reflects the temporal origin and natural history of the underlying lens pathology. Fetal lens fiber cells are not involved in this pathogenic process. Parentheses indicate equatorial axial extent of age-dependent disease-linked Aβpathology in the supranuclear subregion of Down syndrome lenses. See text for details.

Distinctive lens pathology is recognized as a characteristic early-onset ocular phenotype in subjects with DS that may be clinically detectable early in life, and in some cases, at birth [20], [22], [23], [24], [26], [43], [44]. By contrast, DS neuropathology and cognitive sequelae typically emerge in the third decade of life and age-dependently progress thereafter [18], [19], [37], [38], [39], [40]. In the present study, early-onset lens pathology is histochemically documented in illustrative cases of two young adults (21 and 22 years of age) with DS. Lenses obtained from these donors revealed marked Aβ amyloid lens pathology that was evident not only in the supranuclear subregion, but also throughout the anterior and equatorial cortex and epithelium. Aβ biochemical analysis of DS and normal control lenses indicates that increased Aβ expression in the lenses of people with DS may be evident very early in life. In one case included in this study, we detected abnormally high tissue levels of Aβ in the lens of a DS subject at 2 years of age. Although visual impairment may not become clinically apparent due to the peripheral distribution of these lenticular lesions relative to the iris, it is certainly the case that Aβ molecular pathology in the lens may be amongst the earliest expressed age-dependent phenotypes in Down syndrome.

The DS-associated cataract phenotype is not observed in other non-AD neurodegenerative diseases, nor in normal aged controls. In this context, it is important to note that DS-associated cataracts are phenotypically distinct from age-related cataracts (ARC) that commonly emerge starting in the fifth decade of life and typically localize in the central nuclear region. In contrast to the lens pathology in ARC, the DS lens phenotype is often clinically detectable early in life, and in some cases, may be present at birth [20], [22], [23], [24], [26], [43], [44]. The distinctive lens phenotype associated with DS is further distinguished from ARC by supranuclear Aβ histopathology, a pathological feature shared with late-onset AD [28].

The results of the present study support a DS-associated pathogenic pathway linking progressive age-dependent Aβ accumulation in the lens and supranuclear cataractogenesis with corresponding cerebral Aβ accumulation and neuropathology in the brain. We propose the following mechanistic pathway leading to expression of the characteristic lens and brain phenotypes associated with DS (Fig. 9). Increased APP gene dosage resulting from chromosome 21 triplication (APP, 21q21) leads to increased expression of the amyloid precursor protein (APP) and elevated levels of the pathogenic Aβ cleavage peptides in affected tissues. In the brain, Aβ accumulation leads to early-onset neuropathology and progressive age-dependent neurocognitive sequelae. With respect to the lens, the present investigation supports the hypothesis that increased expression of Aβ in the lens leads to amyloidogenic interactions with other structural proteins (e.g., αB-crystallin) within the cytoplasm of supranuclear lens fiber cells. This pathogenic cascade leads to increased lens protein aggregation, light scattering, opacification, and ultimately, disease-linked cataracts [45]. Given the fact that the long-lived lens fiber cells are metabolically sluggish and demonstrate limited capacity to catabolize aggregated protein, we speculate that DS-linked Aβ amyloid pathology in the lens may precede corresponding Aβ pathology in the brain. Regardless, the proposed pathogenic model for Aβ pathology in both tissue compartments (brain and lens) is in general agreement with a broad conception of the amyloid cascade hypothesis [16], [46], [47].

Triplication of human chromosome 21 in Down syndrome results in increased dosage of the APP gene (21q21), overexpression of the Alzheimer's disease amyloid-β precursor protein (APP), and progressive accumulation of amyloidogenic amyloid-β peptides (Aβ) in the brain and lens. Deposition of Aβ in both anatomical compartments results in age-dependent Aβ amyloid pathology and disease-linked tissue-specific phenotypes in both Down syndrome and Alzheimer's disease. See text for details.

We hypothesize that the Aβ detected in DS lenses is generated endogenously. The human lens expresses the full complement of enzymes and precursor proteins necessary to generate and process Aβ [28], [48], [49], [50]. In the present study, this hypothesis is supported by the finding of intense Aβ immunoreactivity in DS lens epithelial cells. Our finding that lens fiber cell Aβ is exclusively cytoplasmic suggests that this amyloidogenic peptide may be released from intracellular processing compartments during organelle disintegration accompanying epithelial-to-fiber cell terminal differentiation. This model points to the lens as an appropriate target for ascertaining the temporal origin and natural history of AD-linked molecular pathology in vivo.

Our findings suggest an apparent temporal discordance in DS with respect to phenotype expression in the lens and brain, with pathology in the former preceding that in the latter. However, we also observed variability in age-dependent phenotypic expression of Aβ molecular pathology in the lens that comports with analogous variability in the brain of subjects with DS [34], [51]. The significance and natural history of variability in Aβ tissue concentration in the lens and its possible relationship to the brain in DS are subjects of ongoing investigation.

In this study, we identify Aβ amyloid pathology as the shared molecular etiology of two defining features of DS, namely, supranuclear cataracts in the lens and AD neuropathology in the brain. The proximal pathogenesis of both pathologies likely derives from the primary chromosomal disorder and associated APP gene-dosage imbalance (Fig. 9). The results reported here support a testable pathogenic mechanism by which the primary chromosomal disorder in DS may be translated into a distinctive phenotype expressed in the lens. In this regard, the phenotypic concordance of Aβ lens pathology in both diseases, DS and AD, is striking. Given that DS-linked pathology may be indicative of aberrant Aβ accumulation in both compartments, the lens may provide an optically accessible target tissue for early detection and longitudinal assessment of early Aβ molecular pathology in DS and AD. Future clinical studies will be required to systematically evaluate the frequency of this supranuclear phenotype in DS patients. Quasi-elastic light scattering instrumentation or other techniques may provide a practical approach for quantitative assessment of specific lens pathology throughout the course of the disease.

Supporting Information

Stereo image pair demonstrating mature supranuclear pathology in the lens (with intact zonule fibers) from a subject with Down syndrome. Characteristic circumferential supranuclear cataract in the lens of a 64-year-old male subject with Down syndrome. This distinctive cataract is evident as an annular half-toroid band of opacification in the deep cortical and supranuclear subregions of the lens (shown with intact zonules). This same lens specimen is presented as a slit lamp image (Fig. 1H) and as a stereo image pair (without zonules, Fig. 2A). This dramatic Down syndrome cataract is phenotypically comparable to the subequatorial supranuclear cataract observed in advanced Alzheimer's disease. These distinctive supranuclear cataracts are not observed in age-normal control subjects. See text for details.

Acknowledgments

We thank Mr. Joseph Bertelsen (Signet Laboratories, Covance, Dedham, MA) for generously providing 6E10 and 4G8 monoclonal antibodies. We gratefully acknowledge the National Disease Registry Interchange, Sun Health Research Institute, and the Florida Lion's Eye Bank for their assistance with tissue procurement. We would like to thank the many patients and families who made this work possible.